Methods for identifying genomic equivalent markers and their use in quantitating cells and polynucleotide sequences therein

Abstract

Methods for identifying genetic sequences useful as genomic equivalent markers for organisms are described. The method involves determining the ratio of the absolute number of copies of wild type and mutant amplicons in a number of samples from organisms heterozygous for the mutation. After establishing the number of copies of a particular genetic sequence per genome, the sequence may be used as a measure of the number of genomes per sample, in order to normalize the analysis of another target sequence to abundance per cell. By way of example, the CCR5 gene was shown to be present at 2 copies per genome, and used to measure the number of copies per cell of HIV-1 provirions, human herpesvirus-8, and alpha deletion circles, a measure of recent thymic emigrants for assessing immune reconstitution. The genomic equivalent marker may be use to identify other genomic equivalent markers based on their copy number in proportion to a previously established marker; by way of example the copy number of the beta-actin gene was found to be 16 copies per genome. The genomic equivalent marker may also be use to determine number of cells in a sample, such as from a tissue sample.

What is claimed is:  
     
       1. A method for determining in a nucleic acid sample of cellular origin the number of copies per cell of at least one preselected polynucleotide target sequence, comprising quantifying in said sample the abundance of said at least one target sequence, quantifying in said sample the abundance of at least one genomic equivalent marker sequence, and expressing said number of copies of said at least one target sequence per cell as a ratio between said abundance of said target sequence and said abundance of said at least one genomic equivalent marker. 
     
     
       2. The method of claim  1  wherein said genomic equivalent marker is selected from the group consisting of CCR5 and β-actin. 
     
     
       3. The method of claim  1  wherein said genomic equivalent marker is CCR5. 
     
     
       4. The method of claim  1  wherein said quantifying of said at least one target sequence and of said at least one genomic equivalent marker sequence is performed using real-time PCR amplification in combination with molecular beacons. 
     
     
       5. The method of claim  4  wherein said at least one genomic equivalent marker is CCR5. 
     
     
       6. The method of claim  5  wherein said quantifying of CCR5 is performed using amplification primers SEQ ID NO:2 and SEQ ID NO:3. 
     
     
       7. The method of claim  5  wherein said quantifying of CCR5 is performed using a molecular beacon with a sequence of SEQ ID NO:4. 
     
     
       8. The method of claim  7  wherein said molecular beacon is Tetramethylrhodamine-GCGCCTATGACAAGCAGCGGCAGGAGGCGC-DABCYL SEQ ID NO:4. 
     
     
       9. The method of claim  1  wherein said target sequence is a marker of thymocyte proliferation. 
     
     
       10. The method of claim  9  wherein said marker of thymocyte proliferation is a T cell receptor gene DNA deletion circle. 
     
     
       11. The method of claim  10  wherein said T cell receptor circle is selected from the group consisting of α1 circles, α2 circles, δ1 circles, δ2 circles, δ3 circles, δ4 circles, δ5 circles, and combinations thereof. 
     
     
       12. The method of claim  10  wherein said T cell receptor circle is selected from the group consisting of α1 circles, α2 circles, and combinations thereof. 
     
     
       13. The method of claim  12  wherein said quantifying of α1 circles is performed using primers SEQ ID NO:6 and SEQ ID NO:7 and a molecular beacon with a sequence of SEQ ID NO:8. 
     
     
       14. The method of claim  13  wherein said molecular beacon is Fluorescein-CGAGGCGAGMCGGTGAATGMGAGCAGACAGCCTCG-DABCYL SEQ ID NO:4. 
     
     
       15. The method of claim  12  wherein said quantifying of α2 circles is performed using primers SEQ ID NO:6 and SEQ ID NO:10 and a molecular beacon with a sequence of SEQ ID NO:8. 
     
     
       16. The method of claim  1  wherein said target sequence is a pathogen. 
     
     
       17. The method of claim  1  wherein said quantification of said at least one target sequence is determined by a method selected from the group consisting of real-time PCR and competitive PCR. 
     
     
       18. The method of claim  1  wherein said quantifying of said at least one target sequence and said quantifying of said at least one genomic equivalent marker are performed simultaneously. 
     
     
       19. The method of claim  1  wherein said at least one target sequence is DNA. 
     
     
       20. A method for determining in a nucleic acid sample of cellular origin the number of copies per cell of at least one preselected polynucleotide target sequence, comprising the steps of: 
       a. providing a nucleic acid sample derived from a sample of cellular origin in which said determining the number of copies per cell is desired;  
       b. providing forward and reverse primers for each of said at least one target sequences and said at least one genomic marker sequence;  
       c. providing a molecular beacon capable of binding to a subsequence within the target sequence for each of said at least one target sequence and said at least one genomic equivalent marker sequence;  
       d. incubating said nucleic acid sample, said primers and said molecular beacons together with the necessary components and under real-time PCR conditions to amplify said at least one target sequence and said at least one genomic equivalent marker sequence and to cause the interaction between said molecular beacons and said sequences present in said sample;  
       e. monitoring the change in fluorescence with time of each of said molecular beacons during said real-time PCR;  
       f. quantitating the abundance of any of said at least one target sequence in said sample and the abundance of said at least one genomic equivalent marker sequence by correlating the threshold cycle of each molecular beacon with a predetermined relationship between the threshold cycle and the quantity of the sequence; and  
       g. expressing the number of copies of said at least one target sequence per cell as the ratio of the abundance of said at least one target sequence with the abundance of said at least one genomic equivalent marker.  
     
     
       21. The method of claim  20  wherein said at least one target sequence is α1 deletion circles and said at least one genomic equivalent marker is CCR5. 
     
     
       22. The method of claim  21  wherein said primers for α1 circles are SEQ ID NO:6 and SEQ ID NO:7, said α1 molecular beacon is Fluorescein-CGAGGCGAGMCGGTGMTGAAGAGCAGACAGCCTCG-DABCYL SEQ ID NO:8, said CCR5 primers are SEQ ID NO: 2 and SEQ ID NO:3, and said CCR5 molecular beacon is Tetramethylrhodamine-GCGCCTATGACAAGCAGCGGCAGGAGGCGC-DABCYL SEQ ID NO:4.